JP7302785B2 - Antiseptic or wound-healing agent for Pseudomonas aeruginosa-infected wounds - Google Patents

Description

The present invention relates to an antiseptic or wound healing agent for Pseudomonas aeruginosa infected wounds.

Pseudomonas aeruginosa is known as a causative bacterium that causes serious infections in hospitals and homes. Antiseptic ingredients such as o-phenylphenol and tert-alkylammonium salts are specifically used to control this Pseudomonas aeruginosa, but even these ingredients are not sufficient to inhibit P. aeruginosa. In view of such problems, for example, in Patent Document 1, (a) at least one naturally occurring substance selected from the group consisting of anacardic acid, limonene, β-pinene, farnesol, β-citronellol, pine resin and hinokitiol and ( b) An anti-pseudomonas aeruginosa agent containing 0.2 to 100 parts by weight of indole per 1 part by weight of the naturally-derived substance as an active ingredient has been proposed.

Prior patent documents

JP-A-5-271073

An object of the present invention is to provide a novel antiseptic agent or wound healing agent for Pseudomonas aeruginosa-infected wounds, which is mainly composed of inorganic materials, unlike conventional organic materials.

The present invention (1) is
a primary firing step of firing a starting material containing calcium carbonate and/or calcium hydroxide to obtain a primary fired product;
A fine pulverization step of finely pulverizing the primary fired product;
A secondary firing step of re-firing the primary fired product to obtain a secondary fired product;
A secondary cooling step of cooling the secondary fired product to the outside temperature in a vacuum atmosphere or an inert gas atmosphere;
It is a disinfectant or wound healing agent for Pseudomonas aeruginosa-infected wounds, characterized by being a calcium oxide-containing baked product obtained by.
The present invention (2) is
The antiseptic or wound healing agent for Pseudomonas aeruginosa-infected wounds according to the invention (1), wherein the starting material is a shell.
The present invention (3) is
The disinfectant or wound healing agent for Pseudomonas aeruginosa-infected wounds according to the invention (2), wherein the shell is a scallop shell or an oyster shell.
The present invention (4) is
It has a dense cocoon-like particle surface structure,
The calcium oxide content measured by differential thermal thermogravimetric analysis (TG-DTA) is 95% by weight or more, and the calcium hydroxide content is 5% by weight or less,
The calcium element content measured by X-ray fluorescence spectroscopy (XRF) is 95 atom% or more,
Calcium oxide content measured by X-ray diffraction analysis (XRD) is 95% by mass or more,
The average particle size is 20 μm or less,
Disinfectant for Pseudomonas aeruginosa-infected wounds, characterized by being a calcium oxide-containing baked product obtained by baking shells having a BET specific surface area of 0.5 m ² /g or more and 3.0 m ² /g or less, or It is a wound healing agent.
The present invention (5) is
The disinfectant or wound healing agent for Pseudomonas aeruginosa-infected wounds according to the invention (4), wherein the shell is a scallop shell or an oyster shell.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel antiseptic or wound healing agent for Pseudomonas aeruginosa-infected wounds, which is mainly composed of an inorganic material, which is different from conventional organic materials.

1 is an SEM photograph of Example 1; The in vitro bactericidal activity of the disinfectant against Pseudomonas aeruginosa in the examples is shown. Here, Pseudomonas aeruginosa was reacted with each concentration of BisCaO suspension, hypochlorous acid water, and povidone-iodine solution for 5 minutes. Viable counts were taken to verify the lowest effective concentration of each disinfectant (N=6). In the examples, the in vivo disinfection activity of the disinfectant against Pseudomonas aeruginosa-infected wounds is shown. Pseudomonas aeruginosa-infected wounds in hairless rats were washed with 3 mL of each antiseptic 5 times daily for the first 3 days while gently rubbing with gauze, and then covered with CNFS. Subsequently, from days 4 to 9, the infected wounds were washed with saline only and covered with CNFS. Before washing with each antiseptic solution (days 1, 2, 3, 6, 9), the number of surviving bacteria was measured by a wiping test using a piece of sterile gauze from the infected wound. Data represent mean±standard deviation (SD) (N=7). In addition, BisCaO in the figure means the suspension according to the example (the same applies hereinafter). 1 shows a digital photograph of an open infected wound in an example. Here, Pseudomonas aeruginosa-infected wounds in hairless rats were washed daily with the respective antiseptic solution or saline and covered with CNFS. On the indicated days, each infected open wound (N=7) before irrigation was photographed and the percent open was measured. Figure 3 shows the percentage of open wounds in the examples. Here, the open wound area on Day 1 was taken as 100, and the percentage of each open wound was calculated using the digital photographs of FIG. Data represent mean±standard deviation (SD) (N=7); ^* P<0.05 (Student's t-test). Fig. 3 shows histological examination of granulation tissue and neovascularization on Day 9 in the Examples. Here, the surrounding skin including the wound site of each disinfectant-washed group was excised on Day 9, stained with hemaxillin and eosin (H&E), and fixed on slides. Granulation tissue formation and angiogenesis in each tissue were assessed by micrograph (×100) observation of the tissue slides (N=7). The solid line indicates the granulation tissue width, and the arrow indicates neovascularization. Pictures presented are representative of each group. These photomicrographs were used to generate Table 2.

The antiseptic or wound healing agent for Pseudomonas aeruginosa-infected wounds according to the present invention contains a specific calcined calcium oxide. Hereinafter, the baked product, the method for producing the baked product, and the use of the baked product (disinfectant or wound healing agent for Pseudomonas aeruginosa-infected wounds) will be described in this order.

≪Baked goods≫
The starting material for producing the calcined product according to the invention is shells. Seashells refer to materials containing calcium carbonate that are formed as outer shells by organisms commonly referred to as shellfish and similar organisms (mostly belonging to the subphylum Stelura).

Shellfish are generally classified into monovalves, bivalves, and snails. Examples of monovalves include abalone and Tokobushi, examples of bivalves include scallops, oysters, clams, clams, short-necked clams, and examples of snails include turban shells, whelks, and snails. Although any mussel shell can be used as a starting material, bivalve shells are preferred due to their ease of cleaning and reduced risk of contamination. Among bivalve shells, scallop shells and oyster shells are more preferred, and scallop shells are particularly preferred.

The average particle diameter of the fired product according to the present invention is preferably 20.0 μm or less, 15.0 μm or less, 10.0 μm or less, 8.0 μm or less, 6.0 μm or less, 5.0 μm or less, or 2.0 μm It is below.

The average particle size of the baked product according to the present invention may be measured using a particle size distribution analyzer. An example of such a device is CILAS (Aisin Nano Technologies Co., Ltd.). Further, the shape and surface structure of the fired product of the present invention can be obtained from an SEM image at any magnification of 2000 to 10000 times.

The ratio of calcium element to the elements measurable by wavelength dispersive X-ray fluorescence spectroscopy (XRF) of the fired product is 90 atom% or more, 91 atom% or more, 92 atom% or more, 93 atom% or more, 94 atom% or more, 95 atom% Above, it may be 96 atom % or more, 97 atom % or more, 98 atom % or more, or 99 atom % or more.

By X-ray fluorescence spectroscopy (XRF), in addition to calcium, trace amounts of contents such as potassium, sulfur, phosphorus, magnesium, sodium, aluminum, silicon, and strontium can also be measured. Carbon and oxygen are not measured by wavelength dispersive X-ray fluorescence spectroscopy (XRF).

RIX3100 (manufactured by Rigaku Denki Kogyo Co., Ltd.) is exemplified as an apparatus for wavelength dispersive X-ray fluorescence spectroscopy (XRF).

The contents of calcium oxide, calcium hydroxide, and calcium carbonate in the baked product according to the present invention are estimated using a differential thermal calorimetric analyzer. By differential thermal calorimetric analysis, the water content is estimated from the weight change around 300°C, the calcium hydroxide content is estimated from the weight change around 350°C, and the calcium carbonate content is estimated from the weight change around 600°C. can.

The weight retention ratio (also expressed as calcium oxide content ratio) at 30 to 1000 ° C. measured by differential thermal calorimetric analysis (TG-DTA) of the baked product according to the present invention is preferably 75.0% by weight or more. , 80.0% by weight or more, 85.0% by weight or more, 90.0% by weight or more, 95.0% by weight or more, 99.0% by weight or more, 99.3% by weight or more, or 99.5% by weight or more is. The weight retention rate is the percentage of the weight at 1000°C to the weight at 30°C.

An apparatus for differential thermal thermogravimetric analysis (TG-DTA) includes, for example, TGA851e (manufactured by Mettler Toledo). Differential thermal thermogravimetric analysis is performed by heating from 30° C. to 1000° C. at a rate of 10° C./min in a nitrogen stream of 100 mL/min.

Purity measured by X-ray diffraction analysis (XRD) of the fired product according to the present invention is preferably 90.0% by mass or more, 92% by mass or more, 94% by mass or more, 96% by mass or more, 98% by mass % or more, 99 mass % or more, or 99.5 mass % or more.

X-ray diffraction analysis (XRD) equipment includes, for example, X'Pert-PRO (Philips).

The BET specific surface area of the fired product according to the present invention is preferably 0.2 m ² /g or more, 0.3 m 2 /g or more, 0.4 m ² /g or more, 0.5 m ² /g or more, 0.5 m ² /g or more, 6 m ² /g or more, 0.7 m ² /g or more, 0.8 m ² /g or more, 0.9 m ² /g or more, or 1.0 m ² /g or more. On the other hand, it is preferably 3.0 m ² /g or less, 2.8 m ² /g or less, 2.6 m ² /g or less, 2.4 m ² /g or less, 2.2 m ² /g or less, or 2.0 m ² /g or less.

An example of an apparatus for analyzing the BET specific surface area is ChemBET3000 manufactured by Quantachrome. The method for measuring the BET specific surface area is not particularly limited, and the measurement may be carried out under commonly used conditions.

When the calcined product according to the present invention is subjected to a hydration reaction with water vapor or the like, the surface changes to micronize crystals due to the formation of calcium hydroxide, and the surface structure changes due to the formation of cracks and pores. occurs. Actually, the calcium oxide content measured by X-ray diffraction analysis (XRD) is 99% or more, and the average particle size is 5 μm or less. Compared to baked shells with a content of 75 to 95%, a calcium hydroxide content of 5 to 20%, and an average particle size of 5 μm or less, hydrophilicity and water suspension properties are poor at the beginning of water addition, and more A large amount of precipitate is produced.

≪Manufacturing method of fired product≫
An example of a method for producing the fired product described above is described below. Naturally, the fired product described above may be produced by a method modified from the following method or by a completely different method.

In the manufacturing method, the following steps (1) to (6) are performed in the order described.
(1) Primary firing step of firing shells,
(2) a step of naturally cooling the fired primary fired product to the ambient temperature;
(3) Impurities are removed from the primary fired product through each filter (air filter, micromist filter, activated carbon filter), and the dry ultrafine pulverization system (nano jetmizer) and/or bag or cyclone dust collector is used to convert it into high-pressure gas. A step of uniform fine pulverization and dust collection while replacing and removing carbon dioxide and water vapor by injecting nitrogen gas or argon gas, which is an inert gas, in addition to dried air,
(4) a secondary firing step of secondary firing the primary fired product;
(5) A step of naturally cooling the secondary fired product to ambient temperature under low pressure conditions of 10 ³ Pa or less and/or under inert gas atmosphere conditions;
(6) Carry out the cooled baked product in a nitrogen gas or argon gas atmosphere with the firing furnace opening and closing door (the outside of the firing furnace opening and closing door is also under an argon gas atmosphere), and fill with vacuum and / or nitrogen gas or argon gas. packaging process

Hereinafter, each step will be described by taking the case of using a scallop shell as an example.

In the present invention, the "outside temperature" means the temperature of the ambient environment in which the firing device (firing furnace) is placed. The temperature of the surrounding environment fluctuates depending on the region and place where the kiln is arranged, as well as the time and season, and it cannot be uniformly defined, but it is less than 100°C, less than 80°C, less than 60°C, or less than 50°C. May be interpreted as temperature.

Step (1) is a step of primary firing of the starting material. During this baking, carbon and hydrogen derived from proteins and the like contained in the starting material are released, and calcium carbonate, which is the main component, is transformed into calcium oxide.

The firing temperature is preferably 1200° C. or higher, 1400° C. or higher, or 1600° C. or higher. By setting the temperature above these temperatures, the organic substances can be sufficiently removed and the purity of calcium oxide is increased. On the other hand, the upper limit of the firing temperature is not particularly limited as long as it is below the melting point of calcium oxide (approximately 2600°C). , or 1500° C. or lower. Of course, throughout the firing process, the firing temperature may be constant or may vary, as long as it remains within the above ranges.

The firing time is preferably 3 hours or longer, 4 hours or longer, or 5 hours or longer. On the other hand, the upper limit of the firing time is preferably 8 hours or less, 7.5 hours or less, 7 hours or less, or 6.5 hours or less.

Step (1) is carried out in an oxygen-containing atmosphere (usually in an air atmosphere) to remove organic matter. Carbon and hydrogen contained in proteins and the like react with oxygen to form carbon dioxide and water, which are liberated from the starting materials.

There is no particular limitation on the rate at which the temperature is raised from the ambient temperature to the previous firing temperature, but it is usually 100 to 500°C/hour, 150 to 450°C/hour, 200 to 400°C/hour, or 250 to 350°C/hour. .

Step (2) is a step of cooling the primary fired product fired in step (1). Instead of actively cooling, the heating is stopped and heat is released to naturally cool to the outside temperature. The time required for step (2) is considered to depend on the temperature of the outside air and the starting material, but is generally 10 hours or more, 15 hours or more, or 20 hours or more.

Step (2) may be performed under any atmosphere. For example, an inert gas (helium, nitrogen gas, etc.) atmosphere or an air atmosphere may be used. Moreover, the atmosphere may be the same as or different from that in step (1). Cooling in a low-humidity environment is preferred to prevent hydration reactions.

It is understood that in the process of slow natural cooling, calcium oxide is cooled while maintaining high crystallinity.

In step (3), impurities are removed from the powdered calcined product through filters such as air filters, micromist filters, activated carbon filters, etc., and the particles are accelerated with high-pressure gas energy that has been extremely dried by a special compressor. It is pulverized using a device capable of achieving ultra-fine pulverization by collision (Nano Jet Mizer; NJ-300-D). As the high-pressure gas, it is possible to use nitrogen gas or argon gas, which is an inert gas, in addition to air obtained by drying the air.

Step (4) is a secondary firing step of further firing the fired product. After firing the primary fired product, it is thought that the ratio of calcium oxide decreases by reacting with water vapor in the atmosphere and carbon dioxide, which is a gas generated by firing. Therefore, re-firing is performed to maintain and improve the purity of calcium oxide.

The baking temperature in the secondary baking step is preferably 600° C. or higher, 700° C. or higher, 800° C. or higher, 850° C. or higher, 900° C. or higher, 950° C. or higher. Firing at these temperatures or higher can sufficiently convert calcium carbonate and calcium hydroxide into calcium oxide. The firing temperature in the secondary firing step is about 2600° C. (melting point of calcium oxide) or less, and usually 1500° C. or less, 1200° C. or less, or 1000° C. or less.

The firing time of the secondary firing step is preferably 1 hour or longer, 1.5 hours or longer, or 2 hours or longer. On the other hand, it is preferably 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, or 3 hours or less from the viewpoint of the load on the firing furnace and the energy cost.

Step (5) is a cooling step after secondary firing. In order to maintain the content of calcium oxide in the secondary fired product, natural cooling is performed under low pressure conditions of 10 ³ Pa or less and/or under inert gas conditions.

In step (6), an inert gas is injected into the firing furnace, and the firing furnace door is opened and closed. In this case, it is preferable to use a sliding door instead of a double-opening door. It is easy to cover the inner state under an inert gas atmosphere. Furthermore, the baked product is vacuum-packaged under this inert gas atmosphere.

The inert gas in the present invention is not particularly limited as long as it is not reactive with calcium oxide, and examples thereof include helium gas, argon gas, nitrogen gas and oxygen gas.

In addition, what was mentioned above is only an example, and for example, step (5) may be executed instead of step (2). Step (5) may be omitted and step (3) and step (6) may be performed successively.

≪Usage of calcined product≫
The disinfectant or wound-healing agent for Pseudomonas aeruginosa-infected wounds according to the present invention includes the baked product. Here, when formulating the disinfectant or wound healing agent for Pseudomonas aeruginosa-infected wounds according to the present invention, known methods can be used without particular limitation. For example, various known dosage forms such as liquids (including water suspensions and oils), dispersants, pastes, powders, granules, and microcapsules can be used depending on the purpose and application. Among these, for example, when formulating as a liquid agent, the baked product may be appropriately dispersed in a solvent. More specifically, for example, the fired product is blended in the liquid agent at a ratio of 0.1 to 99% by weight (preferably 1 to 80% by weight, more preferably 5 to 50% by weight), It should be dispersed.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

≪Production of calcined calcium oxide≫
(Example 1)
The scallop shells were baked at 1450° C. for 6 hours and allowed to cool naturally to ambient temperature. Impurities were removed from this through an air filter, a micromist filter, and an activated carbon filter, and finely pulverized by a dry ultrafine pulverization system (nanojetmizer). After that, it was baked at 950° C. for 2 hours. This secondary fired product was naturally cooled to the outside temperature under low pressure conditions (10 ⁻⁴ Pa or less).

(Example 2)
The scallop shells were baked at 1450° C. for 6 hours and allowed to cool naturally to ambient temperature. Impurities are removed from this through an air filter, micromist filter, and activated carbon filter, and inert gas such as nitrogen gas or argon gas is injected using a dry ultrafine pulverization system (nano jetmizer) to replace and remove carbon dioxide and water vapor. pulverized.

(Example 3)
The scallop shells were baked at 1100° C. for 4 hours and allowed to cool naturally to ambient temperature.

(Average particle size)
The average particle size of each powder was measured using a particle size distribution analyzer (CILAS; Aisin Nano Technologies Co., Ltd.).

(Calcium element content ratio)
The calcium element content of each powder was measured using a fluorescent X-ray analyzer (RIX3100; manufactured by Rigaku Denki Co., Ltd.).

(Calcium oxide content ratio)
The calcium oxide content and calcium hydroxide content of each powder were measured using a differential thermal calorimetric analyzer (TGA851e; Mettler Toledo) and an X-ray diffractometer (X'Pert-PRO (Philips)). .

(BET specific surface area)
The BET specific surface areas of Examples 1 to 3 were measured using ChemBET3000 manufactured by Quantachrome.

(Electron microscope observation)
The fired product was coated with osmium metal using a neo-osmium coater (Neoc-STB; Meiwaforsyth Co., Ltd., Tokyo), and then examined using a field emission scanning electron microscope (JSM-6340F; JEOL Ltd., Tokyo) at a magnification of 3000. , the surface morphology of the dry powder state was analyzed based on the 10000-fold SEM image.

In Examples 1 and 2, a dense cocoon-like surface structure was observed, and the BET specific area was observed to be inversely proportional to the average particle size of the powder. In addition, in the fired products according to Examples 1 and 2, adjacent particles are firmly fused to each other, dense cocoon-like crystals and particles grow, and the reaction interface area is reduced due to clogging of pores. observed (Fig. 1).

(Preparation of disinfectant or wound-healing agent for Pseudomonas aeruginosa-infected wounds according to Example)
Add the baked product (1 g) according to Example 1 to 1 L of pure water and rotate to mix to prepare a disinfectant or wound healing agent for 1000 ppm (0.1% by weight) of Pseudomonas aeruginosa-infected wounds, and within 1 hour used.

(Preparation of disinfectant or wound-healing agent for Pseudomonas aeruginosa-infected wounds according to Comparative Example)
Hypochlorous acid water (500 ppm, pH 6.5) was prepared by adding 1/10 (vol/vol) 0.5% (5000 ppm) sodium hypochlorite (Yoshida Pharmaceutical) to sterilized pure water. . The pH of the 500 ppm hypochlorous acid water was adjusted to 6.5 by adding 1N hydrochloric acid. The concentration of hypochlorous acid water is determined as residual (free) chlorine by ClO (HClO and ClO-) selection test paper (high concentration measurement range: 25-500 ppm, low concentration measurement range: 1-25 ppm, Kyoritsu Physical and Chemical Research Institute Co., Ltd. ), Tokyo).

(In vitro microbicidal activity)
Pseudomonas aeruginosa (ATCC 27853 (American Type Culture Collection (ATCC), Manassas, USA) was stored and stored at −80° C. using Luria-Bertani (LB) broth containing 50% sterile glycerol. The concentration of Pseudomonas aeruginosa was newly increased to 1.0×10 ⁶ colony forming units (CFU/mL), and the suspensions according to the examples with various concentrations, the hypochlorous acid water according to the comparative examples, and the povidone-iodine were used. Each P. aeruginosa suspension was added to a Petri dish (90 x 15 mm) containing Pseudomonas aeruginosa isolate agar (Neogen Ltd., Michigan, USA) and incubated for 15 minutes at room temperature. and cultured for 24 hours at 37° C. After culture, the formed colonies were counted and the in vitro Pseudomonas aeruginosa bactericidal activity of each disinfectant was evaluated.

(In vivo Pseudomonas aeruginosa infected wound cleansing)
Hairless rats (male, 300-350 g) were purchased from SLC Japan. Animals were kept under suitable conditions (26° C., 55% humidity). On Day 0 of this study, rats were placed under general anesthesia by intraperitoneal injection of pentobarbital sodium (Dainippon Sumitomo Pharma), and a circular punch was applied to the back of the rat using a sterile 8 mm dermal punch (Kai) and surgical scissors. Created a total loss. In order to make an infected wound, 100 μL of the frozen Pseudomonas aeruginosa preservation solution (1.0×10 ⁶ CFU/mL) was dropped onto the above total wound, and a chitin nanofiber sheet (CNFS, deacetylated Degree of conversion: 30%, Besquitin W, Nipro) was covered. The rats were returned to their cages and maintained for 24 hours to form an infected wound. After that, each infected wound was carefully washed 5 times with 3 mL of a sterilized gauze of 1 cm square and 3 mL of sterilizing agent or physiological saline (15 mL in total) kept at 35-40 ° C for 3 days from Days 1 to 3. The infected wound was covered with a piece of CNFS. Subsequently, every day for 6 days from Days 4 to 9, the infected wound was carefully washed five times with only 3 mL of physiological saline kept at 35 to 40° C. using a 1 cm square gauze, and the infected wound was covered with a piece of CNFS. On Days 1, 2, 3, 6, and 9, the number of adherent Pseudomonas aeruginosa was measured by a wiping test using a sterile gauze of 1 cm square before and after cleaning the infected wound. The concentration of the resulting Pseudomonas aeruginosa suspension was adjusted by a 10-fold dilution method, spread on a separate agar Petri dish for Pseudomonas aeruginosa, and cultured at 37° C. for 24 hours. After culturing, the generated colonies were counted to evaluate the number of Pseudomonas aeruginosa. By observing the morphology of the cells using a microscope, it was confirmed that almost all the cells generated in the infected wound were Pseudomonas aeruginosa. Furthermore, each infected wound on Days 1, 2, 3, 6, and 9 was digitally photographed to evaluate the wound closure rate. We also ensured that there were no signs of complications such as acute inflammation, abscess formation, or seroma accumulation during the experimental period.

(Histological test)
After wound cleansing on post-wounding Day 9, rats were anesthetized with sodium pentobarbital. Surrounding skin containing infected wounds was subsequently removed for histological examination (N=6). A peripheral skin sample containing each infected wound was fixed with a 10% formaldehyde solution, embedded in paraffin, and then sectioned to a thickness of 4 μm (Daiwakoki Kogyo). The sections were made perpendicular to the wound surface and the anterior-posterior axis, and approximately 10 x 1.5 mm sections were transferred to glass slides and stained with hemaxillin and eosin (H&E). A coverslip was placed and the tissue was evaluated microscopically. In each section (N=8), a microscopic field showing the wound was photographed and the length of granulation tissue and the number of new capillaries with a diameter ≧10 μm or containing 5 or more erythrocytes were counted.

(Statistical analysis)
Results are expressed as mean ± standard deviation (means ± SDs). The probability of significant difference was determined by the paired Student's t-test as a two-tailed test using the statistical software JMP (SAS Institute Inc.).

{result}
(In vitro microbicidal activity)
Bacterial colonies when the suspension according to the example, the hypochlorous acid water according to the comparative example, and the pidone-iodine solution against Pseudomonas aeruginosa are treated with different concentrations of bactericides in pure water and physiological saline This was done by counting (Fig. 2). Pseudomonas aeruginosa was completely sterilized with the suspension of Examples, the hypochlorous acid water of Comparative Examples, and the pidone-iodine solution of 500 ppm or more. The 250 ppm suspension according to the example and the hypochlorous acid water according to the comparative example were completely sterilized, but the pidone-iodine solution according to the comparative example left a slight bacterial colony. Below 125 ppm, the log ₁₀ CFU/mL value increased in a concentration-dependent manner.

(In vivo Pseudomonas aeruginosa infected wound cleansing)
Pseudomonas aeruginosa-infected wounds in hairless rats were treated with 3 mL of a warmed 1000 ppm suspension (pH 12.2) according to the example, 500 ppm hypochlorous acid water (HClO; pH 6.5) according to the comparative example, 1000 ppm Washed with povidone-iodine solution (15 mL total) for the first 3 days with gentle gauze rubbing and then covered with CNFS. Subsequently, every day from Days 4 to 9 for 6 days, the infected wounds were carefully washed five times with only 3 mL of warmed physiological saline, and the infected wounds were covered with pieces of CNFS. The no-wash group did not wash the wounds during the 9-day experimental period and only received a CNFS coating. Before washing with each disinfectant (days 1, 2, 3, 6, 9), the number of surviving bacteria was measured by a wiping test using a piece of sterile gauze from the infected wound (Fig. 3). All experimental animals showed no signs of complications such as acute inflammation at the wound site, abscess formation, seroma accumulation during the 9-day experimental period. Before washing on Day 1, 2.0×10 ⁵ CFU or more of Pseudomonas aeruginosa was detected in the suspension obtained from the infected wound wiping test. After washing the wound with the suspension according to the example, the hypochlorous acid water, the povidone-iodine solution, and the physiological saline according to the comparative example, the number of viable bacteria was about 1.8×10 ³ and 1.1×10 ⁴ , respectively. , 1.2×10 ⁴ , 7.3×10 ⁴ CFU. This result indicates that from the first washing on Day 1, the washing using the suspension according to the example can sterilize bacteria more efficiently than the other groups. On the other hand, it was observed that the number of bacteria slightly increased on Day 1 in the group without any washing. After washing the wound with the suspension according to the example, hypochlorous acid water, povidone-iodine solution, and physiological saline on Day 3, the number of remaining bacteria was about 70, 8.5×10 ³ , 9×10 ^{3 , and 2.5×10 3} , respectively. 5×10 ⁴ CFU. On Day 6, Pseudomonas aeruginosa was completely eradicated from the infected wounds washed with the suspension according to the example for 3 days, but from the infected wounds washed with hypochlorous acid water, povidone-iodine solution, and physiological saline, 7.3. 8×10 ² , 1.8×10 ³ , 2.0×10 ³ CFU were detected with no statistically significant difference among the three groups. Furthermore, on day 9, Pseudomonas aeruginosa was completely eradicated from the infected wounds washed with hypochlorous acid water, povidone-iodine solution, and physiological saline, but 9×10 ³ CFU of residual bacteria were detected from unwashed wounds. rice field. From these results, it was found that washing infected wounds with an antiseptic solution, especially washing with the suspension according to the example, exhibited an effective sterilization effect.

(Healing of Pseudomonas aeruginosa infected wounds by in vivo infected wound cleansing)
The suspension according to the example, the hypochlorous acid water, the povidone-iodine solution, the physiological saline, and the non-washing group according to the comparative example were digital photographs to measure the wound closure rate on days 1, 2, 3, 6, and 9. (Fig. 4). On Days 1 and 2, no significant difference in wound closure was observed in any group. On Days 3-9, significant promotion of wound closure was observed in the wash group of the example compared to the other groups (Fig. 5). On Day 3, the open wound rates of the suspension according to the example, the hypochlorous acid water, the povidone-iodine solution, the physiological saline, and the non-washed group according to the comparative example were 68, 95, 93, 98, and 105%, respectively. rice field. This result revealed that washing with the suspension according to the example applied to the infected wound did not delay wound healing after Day 3, but rather accelerated it.

(Histological test)
Fig. 6 shows micrographs (x 100) of H&E-stained tissue slides for histological examination, day 9, suspension according to Example, hypochlorous acid water according to Comparative Example, povidone-iodine solution, physiological saline Representative photomicrographs of water-washed and non-washed groups. In particular, granulation tissue formation in the non-washed group was suppressed compared to the other washed groups (Fig. 6). Tissue formation was significantly promoted over the non-washed group (Table 2). Regarding the group washed with the suspension according to the example on Day 9, the hypochlorous acid water, the povidone-iodine solution according to the comparative example, and the physiological saline, and the non-washed group, the tissue slide was similarly observed with a micrograph (x 100). Angiogenesis in each tissue was assessed. Angiogenesis at the wound site of each group is indicated by arrows in FIG. As a result, in the non-washed group on day 9, angiogenesis significantly decreased compared to the group washed with the suspension according to the example, the hypochlorous acid water according to the comparative example, the povidone-iodine solution, and the physiological saline. It became clear that The suspension wash group according to the Day 9 example showed the best angiogenesis promotion compared to the other groups.

(Discussion)
In clinical practice, Pseudomonas aeruginosa infection of wounds is a major complication. In an in vitro sterilization test of Pseudomonas aeruginosa, 100 ppm of the suspension according to the example, hypochlorous acid water according to the comparative example, and 500 ppm of povidone-iodine solution completely sterilized Pseudomonas aeruginosa (Fig. 2). . Furthermore, in order to evaluate the disinfection activity and wound healing of Pseudomonas aeruginosa-infected wounds on the back of hairless rats, daily for the first 3 days, the wound was washed with the suspension according to the example (1000 ppm, pH 12.2) and CNFS. , followed by repeated wound washing with physiological saline and coating with CNFS every day from day 4 onwards. As a result, compared with the hypochlorite water, povidone-iodine solution, and physiological saline wash groups according to the comparative examples, the wash group according to the example significantly promoted wound healing as well as disinfection in vivo (Fig. 3, 4). In histological examination, daily wound cleansing with the suspension according to the example for 3 days promoted granulation tissue formation and angiogenesis in infected wounds (Fig. 6, Table 2). In addition, no significant complications were observed in the hairless rats used in the experiment during the initial 3 days of all infected wounds washed with antiseptic solution on each occasion and during days 4-9 thereafter. These results demonstrate that irrigation of infected wounds, limited to the initial 3 days, with suspensions according to the Examples is of clinical use to prevent infection of chronic wounds in failing patients without delaying wound healing. suggesting that there is Antiseptics used in clinical settings, such as hypochlorous acid water and povidone-iodine solution according to comparative examples, are cytotoxic to cells involved in wound healing at high concentrations necessary for bactericidal activity. It has been shown to delay normal wound healing. Therefore, the development of topical antiseptics that reduce bacterial counts without inhibiting wound healing is a challenge in treating chronic wounds.

データは平均値 ± 標準偏差（ＳＤ）を示す。肉芽組織形成では、*Ｐ < ０．０５ｖｓ．ｓａｌｉｎｅ， **Ｐ < ０．０１ ｖｓ．ｎｏ ｃｌｅａｎｓｉｎｇ（ｎ＝７），血管新生では、*Ｐ < ０．０５ｖｓ．ｓａｌｉｎｅ， **Ｐ < ０．０１ ｖｓ．ｎｏ ｃｌｅａｎｓｉｎｇ ａｎｄ ｐｒｏｖｉｄｏｎｅ－ｉｏｄｉｎｅ（ｎ＝７）．

Data represent mean±standard deviation (SD). In granulation tissue formation, *P < 0.05 vs. saline, **P<0.01 vs. *P<0.05 vs. no cleaning (n=7), angiogenesis. saline, **P<0.01 vs. no cleaning and providedone-iodine (n=7).

Claims (4)

A primary firing step of firing a shell containing calcium carbonate and/or calcium hydroxide to obtain a primary fired product;
A fine pulverization step of finely pulverizing the primary fired product;
A secondary firing step of re-firing the primary fired product to obtain a secondary fired product;
A cooling step of cooling the secondary fired product to the outside temperature in a vacuum atmosphere or an inert gas atmosphere;
A method for producing a disinfectant or a wound-healing agent for Pseudomonas aeruginosa -infected wounds, comprising a calcium oxide-containing baked product . 2. The production method according to claim 1 , wherein the shell is a scallop shell or an oyster shell. The calcium oxide content measured by differential thermal thermogravimetric analysis (TG-DTA) is 95% by weight or more, and the calcium hydroxide content is 5% by weight or less,
The calcium element content measured by X-ray fluorescence spectroscopy (XRF) is 95 atom% or more,
Calcium oxide content measured by X-ray diffraction analysis (XRD) is 95% by mass or more,
The average particle size is 20 μm or less,
Disinfectant for Pseudomonas aeruginosa-infected wounds, characterized by being a calcium oxide-containing baked product obtained by baking shells having a BET specific surface area of 0.5 m ² /g or more and 3.0 m ² /g or less, or Wound healing agent. The antiseptic or wound healing agent for Pseudomonas aeruginosa-infected wounds according to claim 3 , wherein the shell is a scallop shell or an oyster shell.

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